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At least 19 recordsLinked to original sources
PubMed1979

Affinity chromatography of Ruta graveolens L. O-methyltransferases. Studies demonstrating the potential of the technique in the mechanistic investigation of O-methyltransferases.

Two discrete furanocoumarin (5- and 8-)O-methyltransferases and a caffeic acid 3-O-methyl-transferase from cell cultures of Ruta graveoleus L. have been copurified by affinity chromatography on 1,6-diaminohexane agarose (AH-Sepharose 4B) linked with S-adenosyl-L-homocysteine (SAH). The furanocoumarin O-methyltransferases, which transfer a methyl group from S-adenosyl-L-methionine (SAM) to the 5- or 8-hydroxyls of linear furanocoumarins, were not retarded by 5-(3-carboxypropanamido)-xanthotoxin (CPAX) immobilized to AH-Sepharose 4B, but addition of SAM to the irrigant buffer led to complete retardation of both enzymes on this affinity system. An analogous phenomenon was observed for the caffeic acid O-methyltransferase, with a ferulic acid ligand coupled to the same insoluble support. SAH was as effective as SAM in promoting binding of the furanocoumarin O-methyltransferases to CPAX and caffeic acid 3-O-methyltransferase to immobilized ferulic acid, respectively. The strong and specific adsorption of these enzymes was abolished by exclusion of SAM or SAH from the irrigant buffer. It is concluded that the enzymes bind first to SAM or SAH, and that this binding process in turn induces the binding site for their specific phenolic substrates or their analogs. Based on these findings, a compulsory-ordered kinetic mechanism for the action of these O-methyltransferases is postulated.

Cells, Cultured↗
PubMed1979

Genetic control of cobalamin binding in normal and mutant cells: assignment of the gene for 5-methyltetrahydrofolate:L-homocysteine S-methyltransferase to human chromosome 1.

When extracts prepared from cultured human or rodent fibroblasts grown in medium containing [(57)Co]cobalamin were analyzed by polyacrylamide gel electrophoresis, most of the intracellular radioactivity migrated with the activity of the cobalamin-dependent enzyme 5-methyltetrahydrofolate:L-homocysteine S-methyltransferase (EC 2.1.1.13). Because the rodent and human forms of this enzyme are electrophoretically different, we used the binding of [(57)Co]cobalamin to detect the presence of the human methyltransferase isozyme in rodent-human somatic cell hybrids. As expected, binding and methyltransferase activities were found to cosegregate, thus confirming genetically their electrophoretic identity. Accordingly, we examined the [(57)Co]cobalamin-binding patterns and human chromosome contents of a panel of 12 rodent-human hybrid clones, and concluded that the gene for the methyltransferase (designated Mtr) is located on human chromosome 1. Using this information, we probed the nature of the molecular defect exhibited by fibroblasts cultured from patients expressing the cbl C mutation. Although these cells are unable to associate newly taken up [(57)Co]cobalamin with the methyltransferase, hybrids of mouse L-cells and cbl C cells containing chromosome 1 show a "reappearance" of the human [(57)Co]cobalamin-methyltransferase. These results indicate that the cbl C mutation does not affect the methyltransferase apoprotein, but rather some metabolic step that must convert cobalamin to a chemical form capable of attaching to the enzyme.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1977

Composition, associated tissue methyltransferase activity, and catabolic end products of transfer RNA from carcinogen-induced hepatoma and normal monkey livers.

This investigation was designed to explore transfer RNA (TRNA) methyltransferase activity, urinary excretion levels of tRNA degradation products, and tRNA base composition in normal monkeys and in those with hepatocellular carcinomas induced by N-nitrosodiethylamine. After the development of the tumor, 24-hr urine specimens were collected, the monkeys were sacrificed, and the livers were removed for tRNA isolation and methyltransferase activity studies. The tRNA methyltransferase activity and capacity and the urinary excretion levels for selected tRNA degradation products (pseudouridine, N2,N2-dimethylguanosine, 1-methylinosine, 7-methylguanine, and beta-aminoisobutyric acid) were elevated for the hepatoma-bearing monkeys when compared to those with normal liver. The isolated tRNA pools were analyzed by high-resolution liquid chromatography, and similar base compositions were found for the hepatoma-bearing and normal monkeys. With the use of methyl-deficient Escherichia coli tRNA as the methyl receptor and the analytical procedure for tRNA anlysis, the methylating ability of the tRNA methyltransferases in hepatoma and normal liver extracts was determined. The hepatoma methyltransferase homogenates were found to produce increased levels of 7-methylguanine, N2,N2-dimethylguanine, and thymine, while the normal liver extracts gave higher levels of N2-methylguanine. These differences were not apparent in the base composition of the tRNA pools. The increased urinary excretion and higher methyltransferase activity of the hepatoma-bearing monkeys without an apparent increase in the methylated base content of their tRNA suggest increased tRNA tf individual isoaccepting tRNA's would be missed by analyzing the tRNA pools. The variations in the individual tRNA methyltransferase activities of the hepatoma and normal liver homogenates indicate a difference in the methlation of their tRNA's.

Animals↗
PubMed2026

Coordinated use of three homocysteine methyltransferases supports l-methionine biosynthesis and environmental adaptation among plant-associated bacteria.

Plant pathogens colonize multiple plant-associated habitats throughout their life cycle, encountering distinct nutrient conditions and microbial communities. l-methionine is required for bacterial growth and environmental adaptation. However, how plant pathogens coordinate l-methionine biosynthetic pathways to adapt to different plant-associated environments remains poorly understood. Here, using the plant pathogen Xanthomonas campestris pv. campestris strain XC1 as a model, we show that three homocysteine methyltransferase pathways allow XC1 to catalyze the final step of l-methionine biosynthesis using different methyl donors and cofactors under different environmental conditions. Bioinformatic and transcriptional analyses identified three homocysteine methyltransferase-associated operons in XC1, mesMXD, mmuPM, and metHRHaHb, corresponding to the MesD-, MmuM-, and MetHaHb-dependent pathways, respectively. MesD uses an endogenously synthesized methyl donor and functions as the dominant homocysteine methyltransferase under l-methionine-limiting conditions, supporting bacterial growth, intracellular l-methionine accumulation, and full virulence. Furthermore, MmuM enables XC1 to use plant-derived S-methylmethionine for l-methionine biosynthesis, whereas MetHaHb enables XC1 to use vitamin B12 supplied by a neighboring bacterium for l-methionine biosynthesis in co-culture. Expression analyses showed that mesMXD was the only homocysteine methyltransferase-associated operon that responded to l-methionine availability, and its expression also decreased when S-methylmethionine- or vitamin B12-dependent pathways supported l-methionine biosynthesis. Comparative genomic analysis further showed that the three-homocysteine methyltransferase configuration is conserved in Xanthomonas and is also present in other plant-associated bacteria. Together, these findings show that a plant pathogen can coordinate endogenous, plant-derived, and microbially supported homocysteine methyltransferase pathways to maintain l-methionine biosynthesis, providing a metabolic strategy for adaptation to plant-associated environments.

Methionine↗
PubMed1979

Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.

Amino Acids↗
PubMed1976

Subcellular localization of S-adenosyl-L-methionine:tRNA methyltransferases with aminoacyl-tRNA synthetases in human and mouse: normal and leukemic leukocytes.

The subcellular distributions of S-adenosyl-L-methionine:tRNA methyltransferases and aminoacyl-tRNA synthetases were investigated with the use of human and mouse normal and leukemic leukocyte cell lines. Differential centrifugation of homogenized cell suspensions produced three pelleted subcellular fractions (nuclear and membrane, microsomal, and postribosomal) and a supernatant fraction. Each fraction was assayed for both methyltransferase activity and synthetase activity. The largest amounts, 40-50%, of total methyltransferase and synthetase activities were localized in either the microsomal or the postribosomal fractions, depending on cell type. In addition, the highest specific activities of these two enzyme systems were found to be present in the microsomal and postribosomal fractions. The psotribosomal fraction from leukemic leukocytes had a methyltransferase specific activity higher than that of the microsomal fraction, while the same two fractions of normal leukocytes had approximately equal activities. Specific activities of aminoacyl-tRNA synthetases were found to be approximately equal for these two fractions, whether they were from normal or leukemic leukocytes. The activity of tRNA methyltransferases and synthetases within the postribosomal fraction of the cytoplasm suggests the existence of high-molecular-weight enzyme complexes for the modification as well as the aminoacylation of tRNA.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Stringent regulation of the synthesis of a transfer ribonucleic acid biosynthetic enzyme: transfer ribonucleic acid(m5U)methyltransferase from Escherichia coli.

This paper describes the regulation of a transfer ribonucleic acid (tRNA) biosynthetic enzyme, the tRNA(m5U)methyltransferase (EC 2.1.1.35). This enzyme catalyzes the formation of 5-methyluridine (m5U, ribothymidine) in all tRNA chains of Escherichia coli. Partial deprivation of charged tRNAVal can be imposed by shifting strains carrying a temperature-sensitive valyl-tRNA ligase from a permissive to a semipermissive temperature. By using two such strains differing only in the allelic state of the relA gene, it was possible to show the tRNA(m5U)methyltransferase to be stringently regulated. Upon partial deprivation of charged tRNAVal, the differential rate of tRNA(m5U)methyltransferase synthesis was found to decrease in a strain with stringent RNA control (relA+), whereas it increased in the strain carrying the relA allele. This increase of accumulation of tRNA(m5U)methyltransferase activity required protein synthesis. Thus, when tRNA is partially uncharged in the cell, the relA gene product influences the expression of tRNA(m5U)methyltransferase gene.

Bacterial Proteins↗
PubMedDate not supplied

Separation of the S-adenosylmethionine: 5- and 8-hydroxyfuranocoumarin O-methyltransferases of Ruta graveolens L. by general ligand affinity chromatography.

Two S-adenosyl-L-methionine:furanocoumarin O-methyltransferases of R. graveolens, acting at the 5- and 8-hydroxyl of the psoralen nucleus, were completely resolved by adsorption on a general affinity ligand, 5-(3-carboxypropanamido) xanthotoxin, followed by specific desorption by bergaptol and xanthotoxol, respectively. The 5-O-methyltransferase was purified 450-fold by this procedure, the 8-O-methyltransferase 112-fold, and both enzyme fractions were electrophoretically homogeneous. No resolution could be achieved of the activity against two 5-hydroxypsoralens or of the activity against two 8-hydroxypsoralens, and conclusive evidence is presented for the existence of only one 5-O-methyltransferase and only one 8-O-methyltransferase acting on linear furanocoumarins.

Chromatography, Affinity↗
PubMed1979

Pineal N-acetyltransferase and hydroxyindole-O-methyltransferase: control by the retinohypothalamic tract and the suprachiasmatic nucleus.

The visual pathway and central neural structures involved in the photic and endogenous regulation of the activity of pineal N-acetyltransferase and hydroxyindole-O-methyltransferase were investigated. The results indicate that the visual pathway regulating both enzymes is the retinohypothalamic tract, and that the inferior accessory optic tract is clearly not involved in the regulation of hydroxyindole-O-methyltransferase activity, as has been previously thought. In addition, the suprachiasmatic nucleus was found to be necessary for the generation of a rhythm in N-acetyltransferase activity in blinded animals, and to be responsible for the tonic elevation of hydroxyindole-O-methyltransferase activity in blinded animals. Finally, it was concluded that the rapid and large daily changes in N-acetyltransferase activity seen in a normal lighting cycle and the much slower and smaller changes in hydroxyindole-O-methyltransferase activity seen only after weeks in constant lighting conditions are mediated by the same neural tract; the different time courses of the effects of environmental lighting may be explained on the basis of different intracellular regulatory mechanisms.

Acetylserotonin O-Methyltransferase↗
PubMed1975

Properties of catechol O-methyltransferases from brain and liver of rat and human.

Kinetic and electrophoretic properties of catechol O-methyltransferases (EC 2.1.1.6) from brain and liver were studied. The enzyme of either rat or human tissues exhibited a single molecular form when subjected to electrophoresis at pH7.9. At pH9 a second, apparently oxidized, form was detected. Isoelectric-focusing experiments also indicated only one enzyme form, which was identical from extracts of brain and liver of each species (pI = 5.2 for rat, 5.5 for human). Similarities between brain and liver catechol O-methyltransferase of a given species were also demonstrated by kinetic parameters, meta/para ratios of products, and inhibitor potencies. Human catechol O-methyltransferase exhibited lower Km values than did the rat enzyme for S-adenosyl-L-methionine, dopamine and dihydroxybenzoic acid. Adrenochrome inhibited both rat and human enzyme. It was concluded (1) that only a single enzyme form could be demonstrated in the physiological pH region; (2) that catechol O-methyltransferase of brain could not be distinguished from the liver enzyme of the same species; and (3) that species differences exist between the enzymes of rat and human tissues.

Adrenochrome↗
PubMed1975

Alteration of glycine N-methyltransferase activity in fetal, adult, and tumor tissues.

Glycine N-methyltransferase activity has been examined in a number of fetal and adult organs, as well as in several rodent hepatomas, using both enzymatic and immunological techniques. In fetal rabbit liver, the activity first appears at a low level at about 20 days postfertilization and rises to high levels after birth, reaching maximum in the adult liver. In fast-growing hepatomas, the activity could not be detected by either enzymatic or immunological assay. It could be detected in the slower-growing hepatomas, but in considerably diminished levels compared with that of normal adult rat liver. Immunoassays gave no evidence for inactive forms of the enzyme in the tissues that had no enzymatic activity. Transfer RNA methyltransferase assays carried out simultaneously showed an inverse relationship to the glycine N-methyltransferase activity. The levels of transfer RNA methyltransferase activity were high in fetal and tumor tissues and lower in normal adult tissues.

Animals↗
PubMed1979

Age-dependent changes in the specificity of tRNA methyltransferases in the cerebellum of the icteric and nonicteric Gunn rat.

The activity of tRNA methyltransferases present in the cerebellum of 6- and 21-day-old nonicteric and icteric Gunn rats was compared using purified E. coli tRNAs as substrates. At 6 days the tRNA methyltransferases of the icteric animals were significantly more effective in methylating tRNAGlu2 and tRNAPhe than were those of their nonicteric counterparts. This relationship reversed itself at 21 days. The action of the tRNA methyltransferases from the 6-day-old icteric animals led to higher proportions of 1-methyladenine in tRNAGlu2 and tRNAPhe than were obtained using the corresponding enzymes of the nonicteric animals. The proportion of N2-methylguanine was also higher, yet only in tRNAfMet and not in tRNAPhe. The study reveals much more extensive fluctuations in the activity and in the substrate recognition specificity among the cerebellar tRNA methyltransferases of the icteric than among those of the nonicteric controls during the crucial 6--21 day period of cerebellar development.

Aging↗
PubMed1976

Effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities in liver microsomes.

The effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities, the two enzymes involved in phosphatidyl choline biosynthesis in liver microsomes, has been investigated. Female rats were fed a 5% ethanol-liquid diet containing amino acids, minerals, vitamins, with and without choline, for 2, 6 and 10 weeks. Control animals were pair-fed the same isocaloric diet with 5% sucrose with and without choline. Ethanol administration with or without dietary choline stimulated significantly (P less than 0.001) the specific activities of phosphatidyl ethanolamine methyltransferase in liver microsomes in the animals fed 5% ethanol for 2, 6, and 10 weeks, when compared to those control animals pair-fed the isocaloric diet with or without choline. Ethanol administration with or without dietary choline for 2 weeks stimulated significantly (P less than 0.02) the specific activities of choline phosphotransferase. The specific activities of phosphatidyl ethanolamine methyltransferase continued to increase in the liver microsomes from the animals in which dietary choline was omitted for 2, 6, and 10 weeks in the sucrose controls and alcohol-fed animals. Ethanol administration stimulates significantly (P less than 0.001) the phosphatidyl ethanolamine methyltransferase specific activities in liver microsomes of animals fed the liquid diet with dietary omission of choline and methionine for 2 weeks.

Alcohol Drinking↗
PubMed1979

An assay for human erythrocyte catechol-O-methyltransferase activity using a catechol estrogen as the substrate.

A radiometric assay for catechol-O-methyltransferase (COMT) activity in human erythrocytes is described that employs 2-hydroxy[3H]estrone, and non-radiolabeled S-adenosylmethionine (SAM) as the cosubstrates. The ease of separation of the product of the reaction, 2-methoxy[3H]estrone from 2-hydroxy[3H]estrone makes it possible to achieve low reaction blanks. The assay is very sensitive, and only 200 microliter of whole blood are used per determination. The assay is highly reproducible. The interassay variability (coefficient of variation) was 6.5% for 24 assays of COMT activity in red blood cells in blood obtained daily for 24 days from one person. In incubations conducted at 37 degrees C for 30 min, the catechol-O-methyltransferase activity was a linear function of enzyme concentration (equivalent to 11 to 180 microliter of packed red blood cells). Employing this assay, we evaluated the catalytic conversion of 2-hydroxyestrone to 2-methoxyestrone by catechol-O-methyltransferase from human red blood cells and found that the apparent Michaelis constant and the apparent maximal rate of reaction were 3 x 10(-7) M and 6.7 x 10(-9) mol . ml-1 erythrocytes . h-1, respectively. The catechol-O-methyltransferase activity measured in erythrocytes obtained from 100 healthy subjects (men and nonpregnant women) was 8.2 +/- 0.17 (mean +/- S.E.) nmol 2-methoxyestrone . ml-1 erythrocytes . h-1.

Carbon Radioisotopes↗
PubMed1978

Identification and properties of two methyltransferases in conversion of phosphatidylethanolamine to phosphatidylcholine.

Two methyltransferases involved in the methylation of phosphatidylethanolamine to form phosphatidylcholine were demonstrated in a microsomal fraction of bovine adrenal medulla. The first methyltransferase catalyzes the methylation of phosphatidylethanolamine to form phosphatidyl-N-monomethylethanolamine. This enzyme has an optimum pH of 6.5, a low Km for S-adenosyl-L-methionine (1.4 micron), and an absolute requirement for Mg2+. The second methyltransferase catalyzes the two successive methylations of phodphatidyl-N-monomethylethanolamine to phosphatidyl-N,N-dimethylethanolamine and phosphatidylcholine. In contrast to the first methyltransferase, it has an optimum pH of 10 and a high Km for S-adenosyl-L-methionine (0.1 mM) and does not require Mg2+.

Adrenal Medulla↗
PubMed1979

Alterations in activity of enzyme catechol-O-methyltransferase in central and peripheral regions of the rat and rabbit during pregnancy, parturition, and the postpartum period.

Natural variations in the activity of the enzyme catechol-O-methyltransferase in central and peripheral regions of the rat and rabbit during pregnancy and parturition were observed. All of the organs of the rat showed a decrease in enzyme activity up to the twenty-first day of pregnancy. Rabbit tissues also demonstrated a decrease in catechol-O-methyltransferase activity during Days 24 to 30 after coitus. Increases in activity of the rat ovary, brain, and hypothalamus took place from during Day 21 after coitus to the onset of parturition. The enzyme activity in the rat hypothalamus reached its maximum level at four hours post partum from the Day 21 value, but the rabbit hypothalamus showed the maximum activity at 0 hour parturition. During post partum hours, rat brain catechol-O-methyltransferase activity was very high but the rabbit brain showed a very low level of activity. Rabbit uterine activity reached its minimum value at Day 31 after coitus, but at the comparable intervals the situation was inversed in the rat. Results show an important modification of monoamine degradation by 3-O-methylation during midgestation in the central and peripheral regions. These modifications may be attributed to the natural variations in endocrine activity taking place during pregnancy.

Adrenal Glands↗